WO2024082178A1 - Récepteur antigénique chimérique bispécifique ciblant cd19 et cd22 - Google Patents

Récepteur antigénique chimérique bispécifique ciblant cd19 et cd22 Download PDF

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WO2024082178A1
WO2024082178A1 PCT/CN2022/126207 CN2022126207W WO2024082178A1 WO 2024082178 A1 WO2024082178 A1 WO 2024082178A1 CN 2022126207 W CN2022126207 W CN 2022126207W WO 2024082178 A1 WO2024082178 A1 WO 2024082178A1
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seq
amino acid
acid sequence
sequence shown
antigen receptor
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PCT/CN2022/126207
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English (en)
Chinese (zh)
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廖雪梅
陈卓
谢曼曼
程晓翠
刘阳
马明柔
浦瞿津
刘婷婷
夏广新
柯樱
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上海医药集团股份有限公司
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Priority to PCT/CN2022/126207 priority Critical patent/WO2024082178A1/fr
Publication of WO2024082178A1 publication Critical patent/WO2024082178A1/fr

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  • the present application relates to the field of biomedicine, and specifically to a chimeric antigen receptor targeting CD19 and CD22 and its use.
  • B-ALL Acute B-lymphocytic leukemia
  • B-ALL Acute B-lymphocytic leukemia
  • the 5-year disease-free survival rate of relapsed or refractory childhood B-ALL patients is 27% and 15%.
  • patients who relapse after allogeneic hematopoietic stem cell transplantation are basically in a hopeless situation.
  • the chemotherapy survival rate is less than 10%, and the overall 20-year disease outcome of B-ALL has not been significantly improved. Therefore, there is a huge clinical need to develop effective treatments for relapsed or refractory B-ALL.
  • single-target CD19 CAR-T or CD22 CAR-T therapies have a high rate of disease recurrence after treatment, with a recurrence rate of about 40%-60%.
  • the main reasons for recurrence are the loss of CD19 antigen or the downregulation of CD19 after targeted immunotherapy and the reduction of cell surface CD22 expression. Therefore, it is urgent to design new CAR-T cells to overcome the shortcomings of currently available CAR-T therapies and improve the treatment effect.
  • the present application provides a bispecific chimeric antigen receptor targeting CD19 and CD22, and a cell comprising the chimeric antigen receptor, which can bind to CD19 and CD22 simultaneously and has a killing effect on target cells.
  • the CD19/CD22 CAR-T therapy provided by the present application is expected to overcome the shortcomings of the CAR-T therapy that has been on the market. Through the dual-targeting design, new recognition targets are added, the possibility of tumor escape through antigen loss is reduced, the probability of disease recurrence after treatment is reduced, and the proportion of memory T cells is increased. It is expected to greatly solve the many defects of existing CAR-T therapies and bring long-term and stable treatment effects to patients.
  • the present application provides a chimeric antigen receptor comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19, and the second antigen binding domain comprises a VHH targeting CD22.
  • first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19
  • second antigen binding domain comprises a VHH targeting CD22.
  • the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the second antigen binding domain comprises at least one CDR in the VHH sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.
  • the present application provides a chimeric antigen receptor, which comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can target CD19, the second antigen binding domain can target CD22, the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the VHH comprises the amino acid sequence shown in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.
  • the first antigen binding domain comprises an antibody or an antigen binding fragment thereof.
  • the second binding domain comprises an antibody or an antigen-binding fragment thereof.
  • the antigen binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the first antigen binding domain is a scFv.
  • the second antigen binding domain is a VHH.
  • the second antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59.
  • the HCDR3 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the second antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58.
  • the HCDR2 of the second antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:12.
  • the second antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57.
  • HCDR1 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:57, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:58, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:59.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:11 or SEQ ID NO:12, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from the group consisting of:
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:19;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:20;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:21.
  • the second antigen binding domain comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:60.
  • H-FR1 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
  • the second antigen binding domain comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:61.
  • H-FR2 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
  • the second antigen binding domain comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:62.
  • H-FR3 of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.
  • the second antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:63.
  • H-FR4 of the second antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:60, H-FR2 comprises the amino acid sequence shown in SEQ ID NO:61, H-FR3 comprises the amino acid sequence shown in SEQ ID NO:62, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:63.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4,
  • the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10
  • the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the second antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from any one of the following groups:
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:8
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:9
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:15
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 16
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23;
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:18
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.
  • the second antigen binding domain comprises VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the VHH of the second antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.
  • the first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:31, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:32, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen binding domain comprises H-FR1, H-FR2, H-FR3 and H-FR4, the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:34, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:35, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:36, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:23.
  • the first antigen binding domain comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:37.
  • the first antigen binding domain comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:38, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:39 (HTS), and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:40.
  • the first antigen binding domain comprises L-FR1, L-FR2, L-FR3 and L-FR4, the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:42, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:43, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:44.
  • the first antigen binding domain comprises VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:45.
  • the first antigen binding domain comprises VH and VL
  • the VH comprises the amino acid sequence shown in SEQ ID NO:37
  • the VL comprises the amino acid sequence shown in SEQ ID NO:45.
  • the VH and VL of the first antigen binding domain are connected by a linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:48.
  • the first antigen binding domain comprises a scFv
  • the scFv comprises the amino acid sequence shown in SEQ ID NO:46.
  • the first antigen binding domain and the second antigen binding domain are directly or indirectly linked.
  • the C-terminus of the first antigen binding domain is directly or indirectly connected to the N-terminus of the second antigen binding domain.
  • the N-terminus of the first antigen binding domain is directly or indirectly connected to the C-terminus of the second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain are connected by a linker.
  • the linker comprises a peptide linker.
  • the linker comprises the amino acid sequence shown in SEQ ID NO:47.
  • the chimeric antigen receptor further comprises a co-stimulatory signal region, wherein the co-stimulatory signal region comprises an intracellular co-stimulatory signal region derived from one or more proteins selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.
  • the co-stimulatory signal region comprises an intracellular co-stimulatory signal region derived from one or more proteins selected from the following group: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM
  • the costimulatory signal region is an intracellular costimulatory signal region derived from 4-1BB.
  • the co-stimulatory signal region comprises the amino acid sequence shown in SEQ ID NO:52.
  • the chimeric antigen receptor includes an intracellular signaling domain, which comprises an intracellular signaling region derived from one or more proteins selected from the following groups: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.
  • an intracellular signaling domain which comprises an intracellular signaling region derived from one or more proteins selected from the following groups: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) L
  • the intracellular signaling domain is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:53.
  • the chimeric antigen receptor comprises a transmembrane region, which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • a transmembrane region which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD8 ⁇ , CD28, 4-1BB, CD4, CD27,
  • the transmembrane region is a transmembrane region derived from CD8.
  • the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:51.
  • the chimeric antigen receptor further comprises a hinge region, which comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • a hinge region which comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region is a hinge region derived from CD8.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:50.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide comprises the amino acid sequence shown in SEQ ID NO:49.
  • the chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, a second antigen binding domain, a linker, a first antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory domain, and an intracellular signaling domain.
  • the chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, a VHH targeting CD22, a linker 1, a VH targeting CD19, a linker 2, a VL targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:55.
  • the present application also provides one or more isolated nucleic acid molecules encoding the chimeric antigen receptor.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell, which comprises the chimeric antigen receptor, the nucleic acid molecule, and/or the vector.
  • the cell comprises an immune cell.
  • the immune cell is selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
  • the cell comprises a T cell.
  • the present application provides a method for preparing the chimeric antigen receptor, the method comprising culturing the cell under conditions allowing the chimeric antigen receptor to be expressed.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable carrier.
  • the present application also provides a method for detecting CD19 and/or CD22, which comprises administering the cells.
  • the present application also provides a kit for detecting CD19 and/or CD22 proteins, which comprises the chimeric antigen receptor and/or the cell.
  • the present application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the vector, and the cell in preparing a drug, wherein the drug is used to prevent and/or treat a disease and/or condition.
  • the diseases and/or conditions include diseases and/or conditions associated with CD19 and/or CD22 expression.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a hematological tumor.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • the present application also provides a method for preventing and/or treating a disease and/or a condition, comprising administering the chimeric antigen receptor, the nucleic acid molecule, the vector, and/or the cell to a subject in need thereof.
  • the diseases and/or conditions include diseases and/or conditions associated with CD19 and/or CD22 expression.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a hematological tumor.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition, which are used for preventing and/or treating diseases and/or disorders.
  • the diseases and/or conditions include diseases and/or conditions associated with CD19 and/or CD22 expression.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a hematological tumor.
  • the tumor is selected from the group consisting of leukemia and lymphoma.
  • Figure 1 shows the binding curve of the CD22 VHH described in this application and human CD22 expressed on the surface of K562 cells.
  • Figure 2 shows the results of flow cytometry detection of the positive rate of CD19/CD22 single and double target CART cells described in this application.
  • Figure 3 shows the results of flow cytometry detection of the affinity between the CD19/CD22 single and double target CART cells and antigens described in this application.
  • FIG4 shows the results of flow cytometry detection of changes in the proportion of TCM cells in the CD19/CD22 single- and double-target CART cells described in this application.
  • Figure 5 shows the killing effect of the CD19/CD22 single- and double-target CART cells described in this application on Nalm6-LUC cells.
  • Figure 6 shows the killing effect of the CD19/CD22 single and double target CART cells described in this application on the Nalm6-LUC single positive 1:1 mixed cell model.
  • Figure 7 shows the killing effect of the CD19/CD22 single and double target CART cells described in this application on the Nalm6-LUC wild type/single positive (8:1:1) mixed cell model.
  • Figure 8 shows the levels of cytokines secreted by the CD19/CD22 single- and double-target CART cells described in the present application after being activated by three types of Nalm6 model target cells.
  • FIG9 shows the fluorescence intensity detection results of in vivo imaging of Naml6-LUC mice.
  • FIG. 10 shows the fluorescence images of the in vivo imaging of Naml6-LUC mice.
  • FIG. 11 shows the results of body weight changes in Naml6-LUC mice.
  • FIG. 12 shows the fluorescence intensity detection results of in vivo imaging of Naml6-MIX-LUC mice.
  • FIG. 13 shows the fluorescence images of the in vivo imaging of Naml6-MIX-LUC mice.
  • FIG. 14 shows the results of body weight changes in Naml6-MIX-LUC mice.
  • single domain antibody or “sdAb” or “VHH” generally refers to a class of antibodies that lack antibody light chains and have only heavy chain variable regions.
  • single domain antibodies can be derived from Bactrian camels, dromedaries, alpacas, llamas, nurse sharks, white sharks or rays (for example, see Kang Xiaozhen et al., Journal of Biotechnology, 2018, 34 (12): 1974-1984).
  • single domain antibodies can be derived from alpacas.
  • Single domain antibodies can be composed of heavy chain variable regions (VH).
  • the term "heavy chain variable region” generally refers to the amino terminal domain of the heavy chain of an antigen binding fragment.
  • the heavy chain variable region can be further distinguished into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • Each heavy chain variable region can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the heavy chain variable region contains a binding domain that interacts with an antigen (eg, CD22).
  • transmembrane domain generally refers to a sequence in a cell surface protein that spans the cell membrane, which may include a hydrophobic alpha helix.
  • the transmembrane domain may be connected to an intracellular signal transduction domain to play a role in transmitting signals.
  • the transmembrane domain may be derived from any type I, type II or type III transmembrane protein.
  • the transmembrane domain can include a transmembrane domain or a combination thereof derived from a histone selected from: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, a subunit of a T cell receptor, a polypeptide constituting a CD3 complex, IL2 receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • a histone selected from: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, a subunit of a T cell receptor, a polypeptide constituting a CD3 complex,
  • the subunit of a T cell receptor can include TCR ⁇ , TCR ⁇ , TCR ⁇ or TCR ⁇ .
  • the polypeptide constituting the CD3 complex may include CD3 ⁇ , CD3 ⁇ , CD3 ⁇ or CD3 ⁇ .
  • the transmembrane domain may include a transmembrane domain derived from the CD8.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor immune cell
  • the CAR can be combined with an immune cell receptor activation intracellular domain based on the antigen (e.g., CD22) specificity of the antibody.
  • Immune cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing the target antigen.
  • CAR and CAR-T cells see, for example, Sadelain M, Brentjens R, Rivi ⁇ ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013; 3(4): 388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. Curr Opin Immunol. 2012; 24(5): 633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev. 2014; 257(1):107-126; and WO2013154760, WO2016014789.
  • the term "costimulatory domain” generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for lymphocytes to effectively respond to antigens.
  • the costimulatory domain can comprise a costimulatory signaling domain or a combination thereof derived from a protein selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86, OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, LFA-1, LIGHT,
  • intracellular signaling domain generally refers to a domain located inside a cell that can transduce a signal.
  • the intracellular signaling domain can conduct a signal into the cell.
  • an intracellular signaling domain is any continuous amino acid sequence used to guide protein targeting.
  • the intracellular signaling domain may include an intracellular signaling domain or a combination thereof derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, CD66d, CD5, CD22, FcR ⁇ , FcR ⁇ , FcR ⁇ , FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP12, and a domain comprising at least one ITAM.
  • the intracellular signaling domain can include an intracellular signaling domain derived from CD3zeta.
  • signal peptide refers to a leader sequence at the amino terminus (N-terminus) of a nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequent surface expression during or after translation.
  • an antibody generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single-chain antibodies (e.g., scFv), and antibody fragments that bind to antigens (e.g., Fab, VHH, Fab' and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens described herein.
  • Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain may be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the term "antigen binding fragment” generally refers to one or more fragments of an antibody that specifically binds to an antigen.
  • the antigen binding function of an antibody can be achieved by a full-length fragment of the antibody.
  • the antigen binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2, or a light chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2.
  • Fab fragment usually a monovalent fragment composed of VL, VH, CL and CH domains;
  • F(ab')2 fragment a bivalent fragment that may include two Fab fragments connected by a disulfide bond at the hinge region;
  • Fd fragment composed of VH and CH domains;
  • Fv fragment composed of VL and VH domains of a single antibody arm;
  • dAb fragment composed of VH domain (Ward et al., (1989) Nature 341: 544-546);
  • the "antigen binding fragment” may also include an immunoglobulin fusion protein, wherein the fusion protein comprises a binding domain selected from the following: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • the antigen binding fragment may also include a single domain antibody.
  • the term "scFv” generally refers to a single-chain antibody, which is an antibody formed by directly connecting the heavy chain variable region and the light chain variable region or connecting them through a connecting molecule (e.g., a connecting peptide).
  • the structure of the scFv from the N-terminus to the C-terminus can be a heavy chain variable region-light chain variable region, a light chain variable region-heavy chain variable region, a heavy chain variable region-connecting peptide-light chain variable region, or a light chain variable region-connecting peptide-heavy chain variable region.
  • the term "monoclonal antibody” generally refers to a group of substantially homologous antibodies, i.e., the individual antibodies comprising the group are identical except for possible naturally occurring mutations present in trace amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • the monoclonal antibodies can be prepared by hybridoma technology or produced in bacteria, eukaryotic animals or plant cells using recombinant DNA methods.
  • Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).
  • humanized antibody generally refers to a chimeric antibody that contains less sequence from non-human immunoglobulin, thereby reducing the immunogenicity of the xenoantibody when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.
  • CDR transplantation Jones et al., Nature 321:522 (1986)
  • its variants can be used.
  • the term "tumor” generally refers to a neoplasm formed by local tissue cell hyperplasia.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to a change in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22" may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with the protein expression of CD22 may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • nucleic acid molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule that can self-replicate in a suitable host.
  • the vector can transfer the inserted nucleic acid molecule into a cell and/or between cells.
  • the vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for the expression of transcription and/or translation of DNA or RNA.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector can produce a desired expression product by culturing a suitable cell containing the vector.
  • the vector may include a lentiviral vector.
  • the term "cell” generally refers to an individual cell, cell line or cell culture that may or has contained a plasmid or vector including a nucleic acid molecule described in the present application, or that can express a chimeric antigen receptor described in the present application or an antigen binding protein described in the present application.
  • the cell may include the offspring of a single cell. Due to natural, accidental or deliberate mutations, the offspring cells may not necessarily be completely identical in morphology or genome to the original parent cell, but can express the chimeric antigen receptor or antigen binding protein described in the present application.
  • the cell can be obtained by in vitro transfection of cells using the vector described in the present application.
  • the cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell).
  • the cell may be an immune cell.
  • the immune cell can be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells.
  • the immune cell can be a T cell.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a subject.
  • the pharmaceutical composition may include the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the cell, and an optional pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such preparations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the term "treat” generally refers to: (i) preventing a disease, disorder and/or condition from occurring in a patient who may be susceptible to the disease, but has not yet been diagnosed with the disease; (ii) inhibiting the disease, disorder or condition, i.e., curbing its development; and (iii) alleviating the disease, disorder or condition, i.e., causing the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition to subside.
  • the present application also includes functional variants, derivatives, analogs, homologues and fragments thereof.
  • a variant of any given sequence refers to a sequence in which a specific sequence of residues (whether amino acids or nucleotide residues) has been modified so that the polypeptide or polynucleotide substantially retains at least one endogenous function.
  • Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, as long as the original functional activity is maintained.
  • the term “derivative” generally refers to any substitution, variation, modification, replacement, deletion and/or addition of one (or more) amino acid residues from/to the polypeptide or polynucleotide of the present application, as long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analog generally refers to a polypeptide or polynucleotide, and includes any mimetic of the polypeptide or polynucleotide, ie, a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.
  • amino acid substitutions such as at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 20 or more) amino acid substitutions, can be made, as long as the modified sequence substantially retains the desired activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogs.
  • proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions may be made based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic properties of the residues, as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing non-electric polar head groups with similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.
  • CD22 generally refers to an important membrane antigen on human B lymphocytes that is associated with proliferation and differentiation.
  • the amino acid sequence of human CD22 can be found in UniProt/Swiss-Prot accession number P20273.
  • the antigen targeted by one of the antigen binding domains of the chimeric antigen receptor (CAR) can be CD22.
  • CD19 generally refers to a leukocyte differentiation antigen on the surface of B cells.
  • the CD19 may include human CD19.
  • the antigen targeted by one of the antigen binding domains of the chimeric antigen receptor may be CD19.
  • the term "about” generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.
  • the present application provides a chimeric antigen receptor comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19, and the second antigen binding domain comprises a VHH targeting CD22.
  • first antigen binding domain comprises a single-chain antibody (scFv) targeting CD19
  • second antigen binding domain comprises a VHH targeting CD22.
  • the present application provides a chimeric antigen receptor, which comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain is capable of targeting CD19, the second antigen binding domain is capable of targeting CD22, the second antigen binding domain comprises at least one CDR in the antibody heavy chain variable region VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO:64.
  • the CDR of an antibody is also called the complementary determining region, which is a part of the variable region.
  • the amino acid residues in this region can contact the antigen or antigen epitope.
  • the antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, North's, Kabat/Chothia, etc. These coding systems are known in the art, and specific references can be made to, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region.
  • the CDR covers the CDR sequence obtained by any CDR division method; it also covers its variants, which include the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids.
  • the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids.
  • the antigen-binding proteins described herein are defined by the IMGT coding system.
  • the second antigen binding domain of the chimeric antigen receptor targets CD22.
  • the second antigen binding domain of the chimeric antigen receptor may comprise an antigen binding protein targeting CD22.
  • the second antigen binding domain of the chimeric antigen receptor may include at least one CDR in the variable region of the antibody heavy chain.
  • the variable region of the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 64.
  • the variable region of the antibody heavy chain may include at least one CDR in the VHH sequence of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
  • the second antigen binding domain of the chimeric antigen receptor may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the second antigen binding domain of the chimeric antigen receptor may comprise an antibody or an antigen binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the second antigen binding domain of the chimeric antigen receptor may be VHH.
  • the HCDR3 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 59.
  • the HCDR3 sequence of the second antigen binding domain may be defined according to the IMGT coding system.
  • the second antigen-binding domain comprises HCDR3, which, compared with the sequence shown in SEQ ID NO: 59, has an amino acid substitution (eg, conservative amino acid substitution, etc.) at an amino acid selected from the following positions: X15 and X17 .
  • ATDPWTDCSLDGRYX 15 YX 17 Y (SEQ ID NO: 59), wherein X 15 can be E or R, and X 17 can be D, G or N.
  • the HCDR3 of the second antigen binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the HCDR2 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 58.
  • the HCDR2 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR2, and the HCDR2 has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at an amino acid selected from the following positions compared to the sequence shown in SEQ ID NO: 58: X 3 .
  • ISX 3 RDGNT (SEQ ID NO: 58), wherein X 3 can be G or S.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12.
  • the HCDR2 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the HCDR1 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 57.
  • the HCDR1 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the antigen binding protein comprises HCDR1, and the HCDR1, compared with the sequence shown in SEQ ID NO: 57, has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at an amino acid selected from the following positions: X3 , X5 and X6 .
  • GFX3VX5X6YA (SEQ ID NO:57), wherein X3 may be P or S, X5 may be A or D, and X6 may be D or G.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • the HCDR1 sequence of the antigen binding protein may be defined according to the IMGT coding system.
  • the second antigen binding domain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:57, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:58, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:59.
  • the second antigen binding domain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:11 or SEQ ID NO:12, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
  • the HCDR1 of the second antigen binding domain may include the amino acid sequence shown in SEQ ID NO: 4, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.
  • the antigen binding protein may include antibody B010-B-22Nb-01 or an antigen binding fragment having the same HCDR3 therewith (e.g., having the same HCDR1-3 therewith).
  • the HCDR1 of the second antigen binding domain may include the amino acid sequence shown in SEQ ID NO:5
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO:12
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the antigen binding protein may include antibody B010-B-22Nb-02 or an antigen binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) therewith.
  • the antigen binding protein may include antibody B010-B-22Nb-03 or an antigen binding fragment having the same HCDR3 (e.g., having the same HCDR1-3) therewith.
  • the HCDR1 of the second antigen binding domain may include the amino acid sequence shown in SEQ ID NO: 6
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 12
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 21.
  • the antigen binding protein may include antibodies B010-B-22Nb-04, B010-B-22Nb-04-H4, B010-B-22Nb-04-H5, B010-B-22Nb-04-H6, or an antigen binding fragment having the same HCDR3 therewith (e.g., having the same HCDR1-3 therewith).
  • the second antigen binding domain of the chimeric antigen receptor may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 60.
  • the H-FR1 of the antigen binding protein has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the following group: X1 and X16 .
  • X 1 VQLVESGGGLVQPGX 16 SLRLSCAAS (SEQ ID NO: 60), wherein X 1 may be A or E, and X 16 may be G or R.
  • H-FR1 of the second antigen binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
  • H-FR2 of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 61.
  • H-FR2 of the second antigen-binding domain has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X1 , X4 , X11 , X12 , X14 , X15 and X17 .
  • X1AWX4RQAPGKX11X12EX14X15SX17 (SEQ ID NO : 61 ) , wherein X1 may be I or M, X4 may be F or V, X11 may be E or G, X12 may be L or R, X14 may be G or W, X15 may be I or V, and X17 may be C or Y.
  • H-FR2 of the second antigen binding domain may comprise the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
  • H-FR3 of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 62.
  • H-FR3 of the second antigen-binding domain has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of X2 , X3 , X7 , X12 , X20 , X21 , X27 , X29 and X30 .
  • YX2X3DSVX7GRFTX12SRDNAKNX20X21YLQMNX27LX29X30EDTAVYYC SEQ ID NO: 62 , wherein X2 may be D or Y, X3 may be A , Q or V, X7 may be E or K, X12 may be I or V, X20 may be S or T, X21 may be L or V, X27 may be D or S, X29 may be E, K or R, and X30 may be D or P.
  • H-FR3 of the second antigen binding domain may include the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.
  • the H-FR4 of the second antigen-binding domain may comprise the amino acid sequence shown in SEQ ID NO: 63.
  • the H-FR4 of the second antigen-binding domain has an amino acid substitution (e.g., conservative amino acid substitution, etc.) at one or more amino acids selected from the group consisting of: X3 and X6 .
  • WGX 3 GTX 6 VTVSS (SEQ ID NO: 63), wherein X 3 can be L or Q, and X 6 can be L or Q.
  • H-FR4 of the second antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the second antigen binding domain may include H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO:60
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO:61
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO:62
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO:63.
  • the second antigen binding domain of the chimeric antigen receptor may include H-FR1, H-FR2, H-FR3 and H-FR4,
  • the H-FR1 may include the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3
  • the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10
  • the H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 13
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-01 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 8
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-02 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 2
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 9
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 15
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-03 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 1
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 16
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 22.
  • the second antigen binding domain may include antibody B010-B-22Nb-04 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 10
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the second antigen binding domain may include antibody B010-B-22Nb-04-H4 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 17
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the second antigen binding domain may include antibody B010-B-22Nb-04-H5 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the second antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 3
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 7
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 18,
  • the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the antigen binding protein may include antibody B010-B-22Nb-04-H6 or an antigen binding fragment thereof having the same H-FR1-4.
  • the second antigen binding domain may comprise a VHH
  • the VHH may comprise the amino acid sequence shown in SEQ ID NO: 64.
  • the VHH may comprise the amino acid sequence shown in any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30.
  • the first antigen binding domain of the chimeric antigen receptor may include an antibody or an antigen binding fragment thereof.
  • the antigen binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the first antigen binding domain of the chimeric antigen receptor may be scFv.
  • the first antigen binding domain of the chimeric antigen receptor can target CD19 protein.
  • the first antigen binding domain of the chimeric antigen receptor may include at least one CDR in the antibody heavy chain variable region.
  • the antibody heavy chain variable region may include the amino acid sequence shown in SEQ ID NO:37.
  • the first antigen binding domain of the chimeric antigen receptor may comprise HCDR3, for example, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen binding domain of the chimeric antigen receptor may comprise HCDR2, for example, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:32.
  • the first antigen binding domain of the chimeric antigen receptor may include HCDR1, for example, the HCDR1 may include the amino acid sequence shown in SEQ ID NO:31.
  • the first antigen binding domain of the chimeric antigen receptor may include HCDR1, HCDR2 and HCDR3.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:31
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:32
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:33.
  • the first antigen binding domain may include H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO:34.
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO:35.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO:36.
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO:23.
  • the first antigen binding domain may comprise an antibody heavy chain variable region VH.
  • the antibody heavy chain variable region VH may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the first antigen binding domain of the chimeric antigen receptor may include at least one CDR in the antibody light chain variable region.
  • the antibody light chain variable region may include the amino acid sequence shown in SEQ ID NO:45.
  • the first antigen binding domain of the chimeric antigen receptor may include LCDR3.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO:40.
  • the first antigen binding domain of the chimeric antigen receptor may include LCDR2.
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 39 (HTS).
  • the first antigen binding domain of the chimeric antigen receptor may comprise LCDR1.
  • the LCDR1 may comprise SEQ ID NO:38.
  • the first antigen binding domain of the chimeric antigen receptor may include LCDR1, LCDR2 and LCDR3.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:38
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:39 (HTS)
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:40.
  • the first antigen binding domain of the chimeric antigen receptor may include L-FR1, L-FR2, L-FR3 and L-FR4.
  • the L-FR1 may include the amino acid sequence shown in SEQ ID NO:41.
  • the L-FR2 may include the amino acid sequence shown in SEQ ID NO:42.
  • the L-FR3 may include the amino acid sequence shown in SEQ ID NO:43.
  • the L-FR4 may include the amino acid sequence shown in SEQ ID NO:44.
  • the first antigen binding domain of the chimeric antigen receptor may comprise an antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:45.
  • the first antigen binding domain of the chimeric antigen receptor may include VH and VL.
  • the VH includes the amino acid sequence shown in SEQ ID NO:37
  • the VL includes the amino acid sequence shown in SEQ ID NO:45.
  • the VH and the VL may be directly or indirectly connected.
  • the VH and the VL may be connected via a linker.
  • the linker may be a peptide linker.
  • the linker may include the amino acid sequence shown in SEQ ID NO:48.
  • the first antigen binding domain of the chimeric antigen receptor may comprise an scFv.
  • the scFv may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the first antigen binding domain may be directly or indirectly connected to the second antigen binding domain.
  • the indirect connection may include connection through a linker.
  • the linker may be a peptide linker.
  • the linker may include the amino acid sequence shown in SEQ ID NO:47.
  • the C-terminus of the first antigen-binding domain may be directly or indirectly connected to the N-terminus of the second antigen-binding domain.
  • the N-terminus of the first antigen-binding domain may be directly or indirectly connected to the C-terminus of the second antigen-binding domain.
  • the chimeric antigen receptor may include a transmembrane domain.
  • the transmembrane domain may include, but is not limited to, a transmembrane domain or a combination thereof derived from a protein selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane domain may include a transmembrane domain derived from CD8.
  • the transmembrane domain may include an amino acid sequence shown in SEQ ID NO:51.
  • the transmembrane domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO:51.
  • the chimeric antigen receptor may include a costimulatory signaling domain.
  • the costimulatory signaling domain may include, but is not limited to, a costimulatory signaling domain or a combination thereof derived from a protein selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.
  • the costimulatory signaling domain may include a costimulatory signaling domain derived from 4-1BB.
  • the costimulatory signaling domain may include an amino acid sequence as shown in SEQ ID NO: 52.
  • the co-stimulatory signaling domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO:52.
  • the chimeric antigen receptor may include an intracellular signaling domain.
  • the intracellular signaling domain may include an intracellular signaling domain or a combination thereof derived from a protein selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • EBV Epstein-Barr virus
  • HSKV Kaposi's sarcoma herpes virus
  • the intracellular signaling domain may include an intracellular signaling domain derived from CD3zeta.
  • the intracellular signaling domain may include an amino acid sequence as shown in SEQ ID NO:53.
  • the intracellular signaling domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence shown in SEQ ID NO:53.
  • the chimeric antigen receptor may include a hinge region.
  • the hinge region includes a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, CD8 ⁇ , PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region is a hinge region derived from CD8.
  • the hinge region may include the amino acid sequence shown in SEQ ID NO: 50.
  • the hinge region may include an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology with the amino acid sequence shown in SEQ ID NO: 50.
  • the chimeric antigen receptor may include a signal peptide.
  • the signal peptide may include an amino acid sequence shown in SEQ ID NO: 49.
  • the C-terminus of the signal peptide may be directly or indirectly connected to the N-terminus of the antigen binding domain.
  • the N-terminus of the transmembrane domain can be connected to the C-terminus of the antigen binding domain.
  • the C-terminus of the transmembrane domain can be connected to the N-terminus of the costimulatory signaling domain.
  • the C-terminus of the costimulatory signaling domain can be connected to the N-terminus of the intracellular signaling domain.
  • the chimeric antigen receptor may comprise the following domains from N-terminus to C-terminus: a signal peptide, a VHH targeting CD22, a linker 1, a VH targeting CD19, a linker 2, a VL targeting CD19, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • the chimeric antigen receptor may include the following domains from N-terminus to C-terminus: a signal peptide, a VH targeting CD19, a linker 2, a VL targeting CD19, a linker 1, a VHH targeting CD22, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 ⁇ intracellular signaling domain.
  • the linker 1 may comprise the amino acid sequence shown in SEQ ID NO: 47.
  • the linker 2 may comprise the amino acid sequence shown in SEQ ID NO: 48.
  • the chimeric antigen receptor may comprise the following domains in sequence from N-terminus to C-terminus: an antigen binding protein of the present application (e.g., VHH), a transmembrane domain derived from CD8, a co-stimulatory signaling domain derived from CD137, and an intracellular signaling domain derived from CD3zeta.
  • an antigen binding protein of the present application e.g., VHH
  • a transmembrane domain derived from CD8 e.g., a transmembrane domain derived from CD8
  • co-stimulatory signaling domain derived from CD137
  • an intracellular signaling domain derived from CD3zeta.
  • the chimeric antigen receptor may comprise the amino acid sequence shown in SEQ ID NO: 55.
  • the chimeric antigen receptor may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology with the amino acid sequence shown in SEQ ID NO: 55.
  • the present application also provides one or more isolated nucleic acid molecules, which can encode the chimeric antigen receptor described in the present application.
  • each of the one or more nucleic acid molecules can encode the complete chimeric antigen receptor, or a portion thereof (e.g., one or more of HCDR1-3 and heavy chain variable region).
  • the nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme cutting and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the chimeric antigen receptor can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the nucleic acid molecule of the chimeric antigen receptor described in the present application.
  • the present application provides one or more vectors, which contain one or more nucleic acid molecules described in the present application.
  • One or more nucleic acid molecules may be included in each vector.
  • other genes may also be included in the vector, such as marker genes that allow the vector to be selected in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host.
  • control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is an adjustable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcription control functionally connected to a nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a certain portion of the promoter may be fused to a certain portion of a promoter of a gene of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not include additional introns.
  • One or more nucleic acid molecules described herein may be operably connected to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector may be an expression vector.
  • the vector may be a viral vector.
  • the viral vector may be administered directly to the patient (in vivo) or may be administered indirectly, for example, by treating cells with a virus in vitro and then administering the treated cells to the patient (ex vivo).
  • Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex virus vectors for gene transfer.
  • gene transfer may be integrated into the host genome using retroviral, lentiviral, and adeno-associated viral methods to allow long-term expression of the inserted gene.
  • a lentiviral vector is a retroviral vector that is capable of transducing or infecting non-dividing cells and typically produces a higher viral titer.
  • the lentiviral vector may comprise a long terminal repeat sequence 5'LTR and a truncated 3'LTR, RRE, a rev response element (cPPT), a central termination sequence (CTS) and/or a post-translational regulatory element (WPRE).
  • the vector described in the present application may be introduced into a cell.
  • the present application provides a kind of cell.
  • the cell may include a chimeric antigen receptor described in the present application, one or more nucleic acid molecules and/or one or more vectors described in the present application.
  • each or each cell may include one or a nucleic acid molecule or vector described in the present application.
  • each or each cell may include multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in the present application may be introduced into the host cell, such as a prokaryotic cell (e.g., a bacterial cell), a CHO cell, a NS/0 cell, a HEK293T cell, a 293F cell or a HEK293A cell, or other eukaryotic cells, such as cells from plants, fungi or yeast cells, etc.
  • the vector described in the present application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.
  • the cell may include a yeast cell.
  • the cell may include an Escherichia coli cell.
  • the cell may include a mammalian cell.
  • the cell may include an immune cell.
  • the cell may include an immune cell.
  • the cell may include an immune cell.
  • the cell may include an immune cell.
  • the cell may include a T cell, a B cell, a natural killer (NK) cell, a macrophage, a NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte and/or a peripheral blood mononuclear cell.
  • the cell may include a T cell.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may include the chimeric antigen receptor described in the present application, the isolated nucleic acid molecule, the vector, the cell, and/or a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex and/or a nonionic surfactant.
  • any conventional medium or reagent may be considered for use in the pharmaceutical composition of the present application.
  • the pharmaceutically acceptable excipient may include an additive other than the main drug in the pharmaceutical preparation, which may also be referred to as an excipient.
  • the excipient may include an adhesive, a filler, a disintegrant, and a lubricant in a tablet.
  • the excipient may include wine, vinegar, and a medicinal juice in a traditional Chinese medicine pill.
  • the excipient may include a matrix part in a semisolid preparation ointment or cream.
  • the excipients may include preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.
  • the present application provides a method for detecting or measuring CD22, which may include using the isolated antigen-binding protein or the polypeptide.
  • the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.
  • the method may include a method for detecting the presence and/or amount of CD22 for non-diagnostic purposes, which may include the following steps:
  • the present application provides a CD22 kit, which may include and use the isolated antigen-binding protein or the polypeptide.
  • the kit may further include instructions for use, which describe methods for detecting the presence and/or content of CD22.
  • the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.
  • the present application provides a use of the isolated antigen-binding protein or the polypeptide in preparing a kit, which can be used for a method for detecting the presence and/or content of CD22.
  • the method may include an in vitro method, an ex vivo method, or a method for non-diagnostic or non-therapeutic purposes.
  • the present application provides an isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, and the pharmaceutical composition for preventing, alleviating and/or treating a disease or condition.
  • the kit and/or the drug combination is used to prevent, alleviate and/or treat a disease or condition.
  • the disease or condition may include a tumor.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to changes in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22” may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with the protein expression of CD22 may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • the present application provides a use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and/or treating a disease or condition.
  • the present application provides a use of a drug combination in the preparation of a medicament for preventing, alleviating and/or treating a disease or condition.
  • the disease or condition may include a tumor.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to changes in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22” may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with CD22 protein expression may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • the present application provides a method for preventing and/or treating a disease or condition, which comprises administering the isolated antigen binding protein, the isolated nucleic acid molecule, the vector, the cell, or the pharmaceutical composition to a subject in need thereof.
  • the disease or condition may include a tumor.
  • the tumor may include a tumor associated with the expression of CD22.
  • the term "tumor associated with the expression of CD22” generally refers to changes in the expression of CD22 in the tumor microenvironment or on the surface of tumor cells compared to normal cells.
  • the "tumor associated with the expression of CD22” may be a tumor in which the expression of CD22 in the tumor microenvironment or on the surface of tumor cells is upregulated compared to normal cells.
  • the tumor associated with CD22 protein expression may be a CD22-positive tumor.
  • the protein expression of CD22 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more compared to normal cells.
  • the tumor may include a blood tumor.
  • the tumor may include a lymphoma.
  • the tumor may include a leukemia.
  • compositions and methods described herein can be used in combination with other types of cancer therapies, such as chemotherapy, surgery, radiation, gene therapy, etc.
  • the pharmaceutical compositions and methods described herein can be used for other disease conditions that rely on immune responses, such as inflammation, immune diseases, and infectious diseases.
  • the subject may include a human or a non-human animal.
  • the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats.
  • the non-human animal may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the human may be Caucasian, African, Asian, Semitic, or other races, or a hybrid of various races.
  • the human may be elderly, adult, adolescent, child or infant.
  • the effective amount in humans can be extrapolated from the effective amount in experimental animals.
  • Freireich et al. described the relationship between animal and human doses (based on milligrams per square meter of body surface) (Freiheim et al., Cancer Chemother. Rep. 50, 219 (1966)).
  • Body surface area can be approximately determined from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
  • a healthy female adult alpaca (Alpaca) was immunized with recombinant human CD22 protein (11958-H02, Beijing Sino Biological Technology Co., Ltd.).
  • 500 ⁇ g of recombinant human CD22 protein was emulsified with an equal volume of Freund's complete adjuvant and injected into the left and right sides near the cervical lymph nodes;
  • 500 ⁇ g of recombinant human CD22 protein was emulsified with an equal volume of Freund's complete or incomplete adjuvant and injected into the left and right sides near the cervical lymph nodes, and several booster immunizations were performed. Blood was collected one week after the last immunization to monitor the antiserum titer.
  • lymphocytes were separated by lymphocyte separation solution; total RNA was extracted by TRIzol TM reagent; cDNA was obtained by reverse transcription using PrimeScript TM II first-strand cDNA synthesis kit, and VHH gene was amplified by nested PCR.
  • the VHH gene fragment was recovered by gel purification kit and digested by restriction endonuclease Bgl 1, then cloned into phagemid vector pADL-10b, and the constructed cloned product was transformed into E.coli TG1 electroporation competent cells to construct VHH gene library; the library capacity was determined to be 2.2 ⁇ 10 9 pfu by plate gradient dilution method, and the colony PCR results showed that the library insertion rate was 97.9%. 10-100 times the library capacity of live cells was taken from the above gene library for inoculation and culture, and M13K07 phage was used for rescue after culture to logarithmic phase. After rescue culture, phage was collected by centrifugation, and PEG-NaCl was used to purify the phage to obtain phage display library, which can be directly used for subsequent screening.
  • the biologically active antibodies that have been screened can be optionally humanized.
  • the humanization of camel-derived monoclonal antibodies is carried out according to the methods disclosed in many documents in the art.
  • the constant domain of the parent can be replaced by the human antibody constant domain, and the human germline antibody sequence is selected according to the homology of the camel-derived antibody and the human antibody, and CDR transplantation is performed.
  • the amino acid residues of VH are reversely mutated, and the constant region of the camel-derived antibody is replaced by the human constant region to obtain the final humanized binding proteins B010-B-22Nb-04-H4, B010-B-22Nb-04-H5 and B010-B-22Nb-04-H6.
  • Table 1 shows the CDR, heavy chain variable region VH, and FR of CD22 VHH of this application.
  • Biacore was used to detect the binding affinity of different CD22 antigen binding proteins to antigen (CD22 protein, source: Beijing Sino Biological Technology Co., Ltd., catalog number: 11958-H08H1-B) protein.
  • CM5 chip 1-8 channels The surface of CM5 chip 1-8 channels was activated with a 1:1 mixture of 50 mM NHS and 200 mM EDC (NHS and EDC were from the amino coupling kit) at a flow rate of 10 ⁇ L/min for 420 seconds.
  • Anti-hFc or anti-mouse Fc antibody (diluted in sodium acetate solution at pH 4.5, concentration 20 ⁇ g/mL) was injected at a flow rate of 10 ⁇ L/min for 200 seconds, and finally the excess active carboxyl groups on the chip were blocked with 1 M ethanolamine hydrochloride (pH 8.5).
  • the chip surface was rinsed with 1 ⁇ HBS-EP+ at a flow rate of 10 ⁇ L/min for 2 hours to stabilize the baseline, and the instrument was set to 25°C.
  • the initial cycle consisting of two steps, sampling and regeneration, was repeated three times before measurement to stabilize the baseline.
  • Sample measurement Inject 1 ⁇ HBS-EP+ buffer into channels 1-8 at a flow rate of 30 ⁇ L/min for 120 seconds and dissociate for 60 seconds.
  • Regeneration Inject 10 mM glycine pH 1.5 into channels 1-8, 30 ⁇ L/min, 30 sec, stabilize for 30 sec.
  • the running buffer for kinetic determination is 1 ⁇ HBS-EP+ (pH7.4) solution.
  • Capture Different antibodies were injected into the test channels of channels 1-8 of the Anti-hFc or anti-mouse Fc chip, respectively, at a flow rate of 10 ⁇ L/min for 60s for capture.
  • the antigen CD22 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 11958-H08H1-B) was diluted to 100nM with 1 ⁇ HBS-EP+ (pH 7.4).
  • Sample measurement Inject into channels 1-8 at a flow rate of 30 ⁇ L/min, and a 0 concentration sample was used to remove background signals; the binding and dissociation times of the antigen and antibody were 180 and 400 seconds, respectively.
  • Regeneration Inject 10mM glycine pH1.5 into channels 1-8 at a flow rate of 30 ⁇ L/min for 30 seconds, and then stabilize for 60 seconds.
  • the equilibrium dissociation constant ( KD value) of each antigen binding protein of the present application was calculated using Biacore8K analysis software.
  • the reference channel (FC1) was used for background subtraction.
  • the binding ability of CD22 antigen binding proteins to human CD22 expressed on the surface of K562 cells was determined based on flow cytometry.
  • the binding ability of different CD22 antigen binding proteins of the present application to human CD22 expressed on the surface of K562 cells was determined by comparing the binding curves.
  • K562 cells were genetically modified to overexpress human CD22, and the cells were named K562-hCD22 cells.
  • K562-hCD22 cells were digested and plated in 96-well plates.
  • the above experiments show that the antigen-binding protein of the present application has binding activity with human CD22 on the surface of K562 cells and is superior to the positive control antibody.
  • the binding curve of the CD22 VHH of the present application and human CD22 on the surface of K562 cells was determined based on flow cytometry.
  • Example 7 CD19/CD22 bispecific CAR molecules and their corresponding single-target specific CAR molecules CD19 CAR, CD22 CAR plasmid construction and lentiviral packaging
  • the gene sequence of the gene-synthesized humanized chimeric antigen receptor molecule targeting CD19/CD22 specifically includes: a single-domain antibody VHH sequence targeting human CD22, Linker1, an antibody VH sequence targeting human CD19, Linker2, an antibody VL sequence targeting human CD19, a membrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3zeta intracellular signal transduction domain, which are connected in series in sequence.
  • the obtained chimeric antigen receptor molecule (CAR molecule) is named B010-B-03 (the amino acid sequence is shown in SEQ ID NO: 55).
  • the gene sequence of the gene-synthesized humanized chimeric antigen receptor molecule targeting CD22 specifically includes: a single-domain antibody VHH sequence targeting human CD22, a membrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3zeta intracellular signal transduction domain, which are connected in series in sequence, and the obtained chimeric antigen receptor molecule (CAR molecule) is named B010-B-22 (the amino acid sequence is shown in SEQ ID NO: 54).
  • the gene sequence of the gene-synthesized humanized chimeric antigen receptor molecule targeting CD19 specifically includes: antibody VH sequence targeting human CD19, Linker2, antibody VL sequence targeting human CD19, membrane domain, 4-1BB co-stimulatory signal transduction domain and CD3zeta intracellular signal transduction domain, which are connected in series in sequence.
  • the obtained chimeric antigen receptor molecule (CAR molecule) is named B010-B-19 (amino acid sequence as shown in SEQ ID NO:56). After synthesis, the chimeric antigen receptor gene sequence is cloned between the EcoRI and BamHI restriction sites on the pLVX-EF1a-IRES-Puro vector.
  • the constructed lentiviral core plasmid and packaging plasmid pMDLg, regulatory plasmid pRSV and envelope protein plasmid pMD2.G are transfected into 293T cells together with PEI transfection reagent, and the virus is collected after a certain period of culture and concentrated for collection.
  • Human peripheral blood T lymphocytes were collected and transfected with the lentivirus prepared in Example 1 to prepare CAR-T cells, which were named B010-B-03, B010-B-19, and B010-B-22 CART, respectively.
  • virus-transfected T lymphocytes obtained by preparing vector packaging using an unrelated sequence were used as the control group and defined as NT-T.
  • the humanized B010-B-03, B010-B-19, B010-B-22 CART cells and NT-T cells infected with the lentivirus in Example 2 were collected and expanded to day 6, day 8, day 10 and day 12.
  • B010-B-03 and B010-B-19 CART can bind to Protein L and CD19
  • B010-B-03 and B010-B-22 CART can bind to CD22.
  • the affinity ratio of Protein L and VHH CAR structure to CD22 protein was used as the CART cell positive rate
  • the binding of CD19 and CD22 was used as the affinity of CART cells to antigens
  • TCM Central Memory T cell, central memory T cell, CD45RO+/CD197+/CD62L+cells
  • the positivity rate and affinity of B010-B-03, B010-B-19, and B010-B-22 CART cells were high and stable, among which the positivity rate and affinity of dual-target CART were not lower than those of single-target CART.
  • the proportion of memory population of dual-target CART showed an upward trend, and the single-target CART reached a peak on Day 8 (Table 3, Figure 2, Figure 3, and Figure 4).
  • Firefly luciferase (Luciferase, abbreviated as LUC) was transferred into Nalm6 to obtain Nalm6-LUC cells.
  • LUC Firefly luciferase
  • Cas9 gene editing knockout technology Cas9 protein and specific sgRNA were transferred into Nalm6-LUC cell lines by electroporation to obtain Nalm6-CD19 - CD22 + -LUC and Nalm6-CD19 + CD22 -- LUC.
  • an in vitro killing assay was performed using an ELISA instrument to compare the killing effects of CART cells on three Nalm6 cell models at different effector-target ratios and compared with NT-T cells.
  • Example 11 Experiment on detecting the level of cytokine secretion by CART cells activated by target cells
  • the CART cells were mixed and co-incubated with the three Nalm6 cell models as the experimental group; the control group was NT-T.
  • B010-B-03 CART was activated to secrete tumor cell-killing-promoting cytokines IL-2, IFN- ⁇ , and TNF- ⁇ at higher levels than B010-B-19 and B010-B-22 CART in the three Nalm6 target cell models (Figure 8).
  • the target tumor cells Nalm6-LUC expressing luciferase constructed by us were inoculated into NSG mice (purchased from Shanghai Southern Model Organisms Technology Co., Ltd.) and injected into the tail vein to form tumors. 2.0 ⁇ 10 5 of the above target cells were resuspended in 200 ⁇ l serum-free culture medium and injected into the tail vein of mice.
  • the luciferase substrate D-Luciferin was injected intraperitoneally into the mouse at a dose of 3 mg/mouse.
  • the mouse was anesthetized and placed in a small animal in vivo imaging instrument for imaging.
  • B010-B-03, B010-B-19, and B010-B-22 CART cells were resuspended in serum-free culture medium and the cell density was adjusted to 2.5 ⁇ 10 7 cells/ml.
  • 5 ⁇ 10 6 CART cells were injected into the tail vein of each mouse, a total of 200 ⁇ l/mouse. Mice treated with non-transfected CAR T cells were set as the control group.
  • regular in vivo imaging tests were performed twice a week to collect the tumor elimination effect.
  • Figure 9 shows the results of in vivo imaging of Nalm6-LUC mice, and the imaging results reflect the number of tumor cells.
  • the results showed that compared with the non-CAR transfected T cell group and the single- and double-target CART cell groups with increasing bioluminescence intensity, the number of tumor cells in the dual-target CART administration group and the single-target CD19 CART administration group was significantly reduced before Day 15, but the CD19 CART administration group showed tumor cell recurrence and growth after Day 15, and the dual-target CART administration group did not recur and grow, and the tumor inhibition rate was 100.0% on the 32nd day after administration. ( Figure 10)
  • FIG11 shows the results of the body weight changes of Nalm6-LUC mice. The results show that the body weight fluctuations of the mice were stable after administration, and no animals died during the treatment period. There was no obvious drug toxicity, and the drug was safe and well tolerated.
  • the cells were detected and identified using a flow cytometer (Beckman Cytoflex). The ratio error was acceptable within ⁇ 10%.
  • Nalm6-MIX-LUC was inoculated into NSG mice (purchased from Shanghai South Model Organisms Science Co., Ltd.) and injected into the tail vein to form tumors. 2.0 ⁇ 10 5 of the above target cells were resuspended in 200 ⁇ l serum-free culture medium and injected into the tail vein of mice.
  • the luciferase substrate D-Luciferin was injected intraperitoneally into the mouse at a dose of 3 mg/mouse.
  • the mouse was anesthetized and placed in a small animal in vivo imaging instrument for imaging.
  • B010-B-03, B010-B-19, and B010-B-22 CART cells were resuspended in serum-free culture medium and the cell density was adjusted to 2.5 ⁇ 10 7 cells/ml.
  • 5 ⁇ 10 6 CART cells were injected into the tail vein of each mouse, a total of 200 ⁇ l/mouse. Mice treated with non-transfected CAR T cells were set as the control group.
  • regular in vivo imaging tests were performed twice a week to collect the tumor elimination effect.
  • Figure 12 shows the results of in vivo imaging of Nalm6-MIX-LUC mice, and the imaging results reflect the number of tumor cells.
  • the results show that compared with the non-CAR transfected T cell group and the single- and double-target CART cell groups with increasing bioluminescence intensity, the number of tumor cells in the dual-target CART administration group was significantly reduced and remained stable without tumor cell recurrence and growth.
  • the tumor inhibition rate was 100.0% on the 32nd day after administration.
  • Figure 14 shows the results of body weight changes in Nalm6-MIX-LUC mice. The results show that the body weight of mice fluctuated stably after administration, and no animals died during the treatment period. There was no obvious drug toxicity, and the drug was safe and well tolerated.

Abstract

L'invention concerne un récepteur antigénique chimérique, comprenant un premier domaine de liaison à l'antigène et un second domaine de liaison à l'antigène, le premier domaine de liaison à l'antigène ciblant CD19, et le second domaine de liaison à l'antigène ciblant CD22. L'invention concerne également une cellule comprenant le récepteur antigénique chimérique, et des procédés de préparation du récepteur antigénique chimérique et de la cellule et des utilisations de ceux-ci.
PCT/CN2022/126207 2022-10-19 2022-10-19 Récepteur antigénique chimérique bispécifique ciblant cd19 et cd22 WO2024082178A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107383196A (zh) * 2017-08-30 2017-11-24 广州百暨基因科技有限公司 人源化抗cd19的抗原结合片段
CN110938148A (zh) * 2018-09-25 2020-03-31 上海恒润达生生物科技有限公司 靶向cd19和cd22嵌合抗原受体
CN112195157A (zh) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 Cd19和cd22双靶点嵌合抗原受体t细胞及其应用
CN113493516A (zh) * 2020-04-02 2021-10-12 重庆精准生物技术有限公司 靶向双特异性位点的嵌合抗原受体及其应用
CN114478803A (zh) * 2022-02-11 2022-05-13 北京大学深圳研究生院 一种新型双特异性嵌合抗原受体的构建及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107383196A (zh) * 2017-08-30 2017-11-24 广州百暨基因科技有限公司 人源化抗cd19的抗原结合片段
CN110938148A (zh) * 2018-09-25 2020-03-31 上海恒润达生生物科技有限公司 靶向cd19和cd22嵌合抗原受体
CN113493516A (zh) * 2020-04-02 2021-10-12 重庆精准生物技术有限公司 靶向双特异性位点的嵌合抗原受体及其应用
CN112195157A (zh) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 Cd19和cd22双靶点嵌合抗原受体t细胞及其应用
CN114478803A (zh) * 2022-02-11 2022-05-13 北京大学深圳研究生院 一种新型双特异性嵌合抗原受体的构建及其应用

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